The present invention relates to ultra sensitive detection methods for biomolecules. Methods for the detection of biomolecules such as nucleic acids, proteins and cellular markers are highly significant not only in identifying specific targets, but also in understanding their basic function. Hybridization probe technologies in particular continue to be one of the most essential elements in the study of gene-related biomolecules. They are useful for a variety of both commercial and scientific applications, including the identification of genetic mutations or single nucleotide polymorphisms (SNP's), medical diagnostics, gene delivery, assessment of gene expression, and drug discovery. Heterogeneous formats for performing such hybridization probe assays have become increasingly common and powerful with the advancement of gene chip and DNA microarray technologies. Such systems allow for high throughput screening of hundreds to thousands of genes in a single experiment. Homogenous formats commonly rely on the output of small molecule fluorescent dyes appended to hybridization probes to indicate the presence or absence of target analyte.
Conjugated polymers (CPs) are efficient light-gathering molecules with properties desirable for a variety of applications. Conjugated polymers can serve as light harvesting materials and signal transducers in fluorescent biosensor applications. These molecules can detect, transduce and/or amplify chemical, biological or physical information into optical and/or electrical signals. Conjugated polymers can provide the advantage of collective response relative to non-interacting small molecule reporters. This collective response influences optoelectronic properties, such as Forster resonance energy transfer (FRET), electrical conductivity and fluorescence efficiency, properties which can be used to report, or “transduce,” target analyte presence.
Water solubility of CPs, a prerequisite for interrogating biological substrates, is usually achieved by charged groups attached to the CP backbone. To date, however, most of the available ionic conjugated polymers are polyanions containing sulfonate or carboxylate functionalities, or polycations containing quarternary amines. Non-charged or neutral groups appended to the CP backbone would be desirable in cases where it is important to reduce or control non-specific electrostatic interactions with biomolecules which typically contain a large number of charged functional groups (amino acids, phosphates, etc.).
Conjugated polymers frequently take the form of rigid-rod structures which have limited flexibility and consequently have a limited ability to adapt to particular three dimensional shapes, thus limiting their ability to conform to the shape of biologically-derived molecules. For example, proteins and nucleic acids, although also polymeric, do not typically form extended-rod structures but rather fold into higher-order three-dimensional shapes to which CPs cannot typically conform.
Majority of the currently available cationic water-soluble conjugated polymers have generally linear “rigid-rod” polymer backbones and therefore experience a limited twist angle between monomer units along the polymer main chain. A consequence of this torsional restriction is that the polymer has a “rigid rod” structure with limited conformations and ability to adapt to the secondary structures of bio-molecules. Additionally, when cationic conjugated polymers are used as light-harvesting molecules, they can deleteriously exhibit fluorescence self-quenching when they cluster near negatively charged biomolecules. Neutral biomolecule/probe conjugates would help address this issue by better isolating the chromophores.
There is a need in the art for novel CPs, for methods of making and using them, and for compositions and articles of manufacture comprising such compounds.